Proteolytic enzymes, or proteases, are proteins that catalyze the degradation of peptide bonds in protein and peptide substrates. Proteases are typically categorized into four major classes (i.e., serine, aspartyl, metallo, and cysteine), classified according to the catalytic site chemical group that facilitates peptide bond hydrolysis. Proteases are involved in a wide variety of physiological and pathological processes including blood coagulation, protein turnover, complement activation, hormone processing, and cancer cell invasion.
Cysteine proteases, for example, are utilized by living organisms to perform a variety of key cellular functions, and thus are potential targets for drug discovery. For example, cathepsin B has been studied for its role in the progression of normal tissue to cancerous tissue, and the protease cruzain is believed to be essential for the parasitic infection in Chagas' disease (a major public health problem in South and Central America, affecting about 25% of the population of those regions).
The interaction of a protease with a substrate is a highly specific binding event that is driven by, for example, favorable molecular shape recognition (i.e., between the protease and the substrate) and electrostatic (e.g., charge-charge, dipolar, or van der Waals) interactions that occur upon binding. Recognition and binding typically involves 3 to 4 amino acid residues of the substrate on either side of an enzyme's catalytic site. Although the kinetics of all proteolytic events are not fully understood, most protease-mediated catalysis occurs because the catalytic site stabilizes a transition-state, structural intermediate in the pathway to peptide-bond cleavage.
Inhibitors of proteolytic activity typically interact with a protease at its active site, preventing interaction (e.g., recognition, binding, or reaction) of enzyme and substrate. However, inhibition via allosteric change (i.e., conformational or other structural change) and co-factor binding inhibition are some other possible modes of inhibition. Potent and specific synthetic inhibitors can: 1) interact with the enzyme's binding pocket with high affinity, and 2) interact with the catalytic site to mimic the transition state structure.
Modulation, e.g., inhibition or enhancement, of protease activity can profoundly influence biological systems, and, therefore, proteases are often chosen as targets for drug discovery. In the design of protease inhibitors, researchers have generally identified chemical structures that interact with the catalytic chemical group at the active site of the protease and find structures that mimic the transition state of the catalytic reaction. These identified structures are then linked to a di- or tri-peptide sequence that specifically binds to the active site substrate binding pockets. Peptide-based inhibitors, mimicking the primary sequence of the natural substrate, often show very high potency against the target; however, orally administered peptides generally exhibit poor bioavailability due to hydrolysis by nonspecific proteolytic enzymes in the digestive system. Substitution of the peptide portion of protease inhibitors with small organic molecules that mimic the molecular shape and charge interactions of the peptides frequently results in improved bioavailability and oral absorption for that inhibitor.